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Team:Bielefeld-Germany/Results/S-Layer/Guide/4a
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Latest revision as of 02:48, 29 October 2011
Small scale cell disruption
The S-layer fusion proteins form inclusion bodies in the E. coli cells (at least most of them). Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. But inclusion bodies are unsoluble so they have to be solubilized by urea or guanidin hydrochloride. In addition, these chemicals suppress the self-assembly ability of the S-layer proteins which leeds to monomeric S-layer proteins. The cell disruption is carried out in a buffer containing 6 M urea, 50 mM phosphate buffer (pH 7.4), 300 mM sodium chloride and 10 mM imidazol. The cells are lyzed by sonification (5 times 2 minutes on ice, max. 20 W, cooling on ice between these steps). Afterwards the lysate is incubated for 1 h at 4 °C on a shaker or vertical rotator to solubilize the inclusion bodies. The cell debris is removed by centrifugation and the supernatant is used for further purification.
Only purified S-layer proteins will self-assemble - click here for further purification steps.
